Women undergoing cardiovascular procedures have a greater perioperative stroke risk than men. Anesthesia for such cardiovascular procedures is an important consideration since anesthetic agents affect ischemic outcome in experimental stroke and may attenuate perioperative stroke incidence. Such anesthetic preconditioning (ARC) may therefore prevent or even delay neurological complications from perioperative stroke. Our preliminary data suggests that the ARC neuroprotection observed in male animals does not occur in females and that ARC in females actually enhances ischemic damage after middle cerebral artery occlusion. This application will focus on how female gender and estradiol-specific effects lead to a detrimental response in ARC female ischemic brain using a mouse isoflurane preconditioning (IsoPC) model. In Aim 1, we will examine the effects of gender and estradiol on ischemic histological and functional outcomes in IsoPC brain. In Aim 2, we will determine if IsoPC neuroprotection in males is mediated by Akt activation and if estradiol increases expression of neuronal cell death-inducible putative kinase (NIPK), a negative modulator of Akt, thus leading to decreased Akt activation in IsoPC females. These novel studies will yield new information about Akt regulation in IsoPC brain. In Aim 3, we will utilize female estrogen receptor (ER) alpha knockout mice and a selective ER alpha agonist to determine if estradiol acts via an ER alpha-dependent mechanism to attenuate Akt activation and increase NIPK gene expression in IsoPC mouse brain, thus leading to increased ischemic damage. In Aim 4, we hypothesize that IsoPC suppresses neuronal nitric oxide synthase (nNOS) activity and consequent peroxynitrite (ONOO) formation, and that the sexually dimorphic response to nNOS-derived nitric oxide cell-mediated death results in reduced infarct injury in IsoPC males and enhanced ischemic damage in IsoPC females. We will also demonstrate that loss of nNOS and reduced ONOO formation leads to greater superoxide-mediated injury in IsoPC female brain. Our findings will elucidate the mechanisms by which female stroke outcomes are worsened by preischemic isoflurane exposure.